title: An Amplex Red-based fluorometric and spectrophotometric assay for L-asparaginase using its natural substrate.
abstract: We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.
authors.affiliation: Max Planck Institute for Dynamics and Self-Organization, D-37077 Goettingen, Germany.
authors.forename: Christos S
authors.initials: CS
authors.lastname: Karamitros
authors.forename: Jiseok
authors.initials: J
authors.lastname: Lim
authors.forename: Manfred
authors.initials: M
authors.lastname: Konrad
mesh_terms: D001215:Asparaginase / Q000378:metabolism*; D057075:Enzyme Assays; D005470:Fluorometry; D006801:Humans; D010078:Oxazines / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D010078:Oxazines; C470430:Amplex Red; C014180:resorufin; D001215:Asparaginase
keywords: Amplex Red; Coupled enzyme assay; Leukemia; l-Asparaginase; l-Aspartate oxidase